Evidence for an Essential Histidine Residue on Active Site of Human Urinary DNase I: Carboxymethylation and Carbethoxylation

Human urinary DNase I was inactivated by monoiodoacetate and monobromoacetate. The inactivation was greater at pH 7.2 than at 6.0 and proceeded in the presence of Ca2+. Amino acid analysis of monobromoacetate-inactivated human urinary DNase I indicated that one histidine residue per mole of the enzyme reacted with monobromoacetate. Diethylpyrocarbonate also inactivated the enzyme, which was protected by DNA in the presence of Mg2+. However, oligonucleotides did not prevent the inactivation even in the presence of Mg2+. Hydroxylamine almost completely restored the activity of the inactivated enzyme by DEP. One histidine residue per mole of the enzyme was calculated to be modified, as shown by the difference spectra of DEP-inactivated enzyme. This histidine residue seems to react with the substrate. These results provide evidence that human urinary DNase I possesses one essential histidine residue at the active site.